Establishment Of Immuno-PCR Methods For Detecting Bordetella Avium And Study On Its Subuit Vaccine

Bordetella avium (B. avium) was first isolated from young turkeys in1967. The disease(Bordetellosis) caused by this bacillus was a kind of height contacted infective epidemicdisease. It mainly causes respiratory diseases, including ophthalmia and rhinitis, in poultries.It has high upper respiratory tracts to keep in touch, the chicks in age of one week can easilyinfected. Bordetellosis in poultry is clinically characterized by death of chicken embryos, lowhatchability, rapid chick death, and ophthalmia in adult chickens. In China, Professor Ruiliang Zhu reported bordetellosis for the first time in1991, then he isolated B.avium fromdiseased embryos and chicks. Bordetellosis undergoes both vertical and horizontaltransmission, so the danger of this disease can be enlarged constantly. In recent years,investigate poultry’s infection rate in the national different areas is between10%and50%byepidemiology. This has already caused the enormous economic losses to raising the poultryindustry of our country. So fast sensitive of diagnostic method and preparation of vaccineseems particularly important to treat in time.This research is made up of five parts:1. Analysis of the antigenic epitope recognized by three monoclonalantibodies against Bordetella avium OMPsIn this experiment, hybridomas (3G10,4A3and4E8) preserved in our laboratory weresubcloned for three rounds using the limiting dilution method. Then prepared ascites. IndirectELISA test proved that three hybridoma cell lines after recovery can secrete antibodiessteadily and the antibodies of ascites have high titres. Antibody titers in the hybridoma cellculture fluid were1:3201:640, and the titers in ascitic fluid were1:64001:12800,respectively. Determine the saturated working concentration of monoclonal antibody, anddraw the saturation curves of monoclonal antibody. According to the saturation curves, thesaturated concentration of3G10,4A3,4E8were1:80,1:801:160, respectively. Then analyzeantigen binding sites of monoclonal antibody through the ELISA additivity test analysis.Calculated additive index AI,3G10,4A3and4E8were all smaller than50%. It was provedthat three strains of monoclonal antibodies recognize the antigen sites may be the same orrelatively close. In western blot analysis, all mAbs recognized a58kDa protein band. In thisexperiment, the analysis of the antigenic sites recognized by monoclonal antibodies, laid the foundation for the development of specific diagnostic reagents.2. Establishment of immuno-PCR methods for detecting Bordetella aviumWe choosed the strain of4E8which has the highest antibody titre as the coated antibody.An ELISA microplate was coated with4E8in carbonate buffer. Rabbit polyclonal antibodiesagainst B. avium were used as detection antibodies. pUC19plasmid as a template, withbiotin-labeled primers for PCR amplification,we prepared the biotin-reporter DNA. Thebiotin-reporter DNA was connected to the biotin-labeled goat anti-rabbit IgG by streptavidinas the linker. Using PCR amplification reports DNA molecule, then, specific amplified bandswere observed after agarose gel electrophoresis. If there is a specific amplified band, it isproved that the detected sample contained B. avium.In this experiment，we determined optimization concentration of the reporter DNA andthe streptavidin. The optimization concentration of the reporter DNA is10ng/mL. Theoptimization concentration of the streptavidin is10ng/mL. Compared the traditional indirectELISA method to the immune PCR with sensitivity, the results was the sensitivity of theindirect ELISA was103CFU/mL, but the immune PCR is1CFU/mL. So, the sensitivity of theimmune PCR method in this test has raised nearly104times. B. avium, chicken P. mirabilis, E.coli, Bordetella bronchiseptica, Pseudomonas aeruginosa and Salmonella were used in the immunePCR to determine the specificity. The results shows that the immune PCR has highlyspecificity.3. Application of immuno-PCR methods for detecting Bordetella aviumTwenty-two strains of Bordetella avium which were preserved in our laboratory weredetected by immuno-PCR. All strains were amplified specific bands. The result showed thatthis method can be used for the detection of Bordetella avian.Samples collected from diseased chicken were detected in this experiment. Theconventional bacterial isolates culture and identification need3or4days to identify thebacterial. It is time-consuming and laborious. Immuno-PCR method can specifically detectavian Bordetella infection, and only need about20hours.Detection of130serum samples collected from diseased chicken were used the method ofplate agglutination test, indirect ELISA and immune PCR. Then we compared this threemethods according to their results. Plate agglutination test resulted of82serum samples werepositive and25equivocal. The positive rate was63.1%; Indirect ELISA test resulted of96serum samples were positive and the positive rate was73.8%; The results of Immuno-PCRshowed that all positive sera detected by the other two methods showed positive and also detected positive sera from suspicious or negative samples. The positive rate was80.7%. Sothe immuno-PCR is more sensitive and has a higher detection rate than the other twomethods.Colonization time of Bordetella avium after infected chicken were detected byimmuno-PCR. The result showed that the trachea and lungs were amplified specific bands byPCR one day after infection; the heart, liver and spleen were amplified specific bands4daysafter infection; the kidney were amplified specific bands6days after infection.4. Comparision of antigenicity and immunogenicity of Bordetella aviumstrainsAfter rejuvenation of22strains, we prepared Bordetella avium hyperimmune serum byimmunizing animals.The antibody titre of each strain was detected by agglutination test. Theresult showed the antigenicity of each strain. According to the different serotypes andgenotypes, we choosed8strains of Bordetella avium which had higher antibody titre. Weanalyzed the existence of a common antigen component between8strains bycross-agglutination test and immune agar diffusion test. Then we analyzed cross-protectiveimmunity among the8strains by protective immunity test.Agglutination test results showed that the22strains of serum antibody titer was between1:320and1280. It proved that all strains can stimulate the body to produce high titerantibodies. The result of cross-agglutination test and immune agar diffusion test showed thatthere was common antigen component between8strains. But cross agglutination antibodytiter is lower than the agglutination test.Immune protection test results showed that the vaccine had a higher immune attack rate ofprotection on the corresponding strain. There is cross-protective immunity when attacked byother strains within the same experimental group, but the rate of protection was lower. TheBa15strain vaccine prepared for the same type of strain or atypical strains attack, showed ahigher immune protection, Ba11and Ba8strains followed.5. Study on subuit vaccine of Bordetella aviumIn this experiment, the omps of Bordetella avium were used as immunogen, and pinepollen polysaccharide, propolis and shortening were used as immune adjuvant. Pine pollenpolysaccharide designed to three doses of10mg/mL,20mg/mL and40mg/mL. Differentimmune adjuvant subunit vaccine of Bordetella avium were prepared by different doses ofpine pollen polysaccharide, propolis and white with the same amount of Bordetella aviumomps. The preparaed vaccines were detected by sterility and safety test. The result showed that they were aseptic and securityPrepared subunit vaccine were used to vaccinate chickens, then detected the immunehumoral and cellular immune indicators of chicks at different times after immunized. Thelevel of antibody in the serum can reflect the state of the humoral immune. The ratio of bloodlymphocytes, lymphocyte subsets in blood IL-2, IFN-content and spleen lymphocytetransformation rate that reflects the level of cell-mediated immunity. The results showed thateach group vaccine can stimulate the body to produce different degrees of antibody levels andpersistent after immunization. Description for the determination of the cellular immuneindicators, the preparation of subunit vaccines were possible to improve the level of immunecells of the immunized animal. In the comparison of various adjuvants, the test results showedthat the indicators of the Group III (40mg/ml pine pollen polysaccharide) was significantlyhigher than the other groups (P <0.05). Group IV (Propolis adjuvant) were higher I (10mg/mlpine pollen polysaccharide), II (20mg/ml pine pollen polysaccharide), V (mineral oil adjuvant)group, but the difference was not significant. Protective immunity test results showed thatGroup III (40mg/ml pine pollen polysaccharide) had the highest rates of protection, and groupIV (propolis adjuvant) had better protection, higher than I, II, and V group. Different adjuvantgroup of immune indicator and immune protection test protection rates were parallel. Itproved that40mg/ml pine pollen polysaccharide can be used as adjuvant of Bordetella aviumsubunit vaccine for the prevention of Bordetella avium infection.In a word, the results of this study showed that three Bordetella avium omps monoclonalantibodies which were preserved in our laboratory can specifically recognize58kD antigenicsites of Bordetella avium omps. They recognized at the same location or adjacent. Weestablished double antibody sandwich immuno-PCR method by4E8strain and polyclonalantibody of Bordetella avium. Experiments showed that this method had high sensitivity andspecificity. In the clinical application, the method was better than traditional testing methodsin the detection time and detection rate. And the immuno-PCR can detect theearly infection ofBordetella avium. According to the antigenic and immunogenic differences analysis of Bordetella avium,we choosed two strain to prepare Bordetella avium subunit vaccine. The results proved that40mg/ml pine pollen polysaccharide can be used as adjuvant of Bordetella avium subunitvaccine for the prevention of Bordetella avium infection.